Fluorescence is a powerful tool for biological analysis, but background interference can necessitate extensive purification for in vitro analysis and can severely constrain in vivo studies. Our goal is to advance biomolecular diagnosis and imaging using small silver clusters comprised of ~10 atoms with strong emission in the near-infrared spectral region where biological samples are relatively transparent. These metallic ligands associate with oligonucleotide sensors that integrate two distinct functions: the formation of specific silver clusters via nucleobase coordination within prescribed sequences and the recognition of target oligonucleotides through complementary base pairing. Confident and highly sensitive detection is accomplished when hybridization of the analyte with this sensor transforms a cluster from a nonemissive state with a violet absorption to a highly emissive state with near-infrared absorption. These sensors are distinguished by their strong and photostable fluorescence response and their economical, convenient, and modular synthesis, thus opening this methodology to a diversity of applications. Our studies will focus on a range of microRNA sequences that are biomarkers for human diseases. The following synthetic, structural, and stability studies will provide the foundation to develop the full potential of this new sensing strategy. I. Optimizing reaction conditions and the sequence environments are critical steps towards forming and transforming specific silver clusters. Highly parallel sequence variations in conjunction with spectral analysis will be coupled with measurements of the cluster stoichiometry and oligonucleotide shape to design sensors that are appropriate for direct analysis in biological samples. II. These metallic chromophores are not only distinguished by their spectra but also by their active role in shaping the sensors. We have shown that a violet absorbing cluster folds its DNA hosts, and biochemical/chemical probes and fluorescent base analogs will be used to map the secondary structure of DNA hosts through solvent exposure of their nucleobases. By identifying where the clusters bind and how they impact the DNA structure, generalized sensors that incorporate cluster-induced folding will be developed. III. Because the cluster coordinates and folds its DNA hosts, hybridization of the target oligonucleotide is inhibited. This impediment will be exploited to make fine distinctions between oligonucleotides that have mismatched base pairs and thus have reduced affinities for a specific sensor. The spectral changes that accompany temperature induced unfolding will provide the quantitative thermodynamic basis for distinguishing such targets. With the structural and thermodynamic insight provided by the above studies, a novel signal amplification scheme based on sequential unfolding will be developed for the detection of low abundance species.